Methods of modulating cytokinin related processes in a plant using B3 domain proteins

ABSTRACT

The present invention is directed to plant genetic engineering. In particular, it relates to methods of modulating cytokinin related processes in a plant and selecting a plant having a phenotype associated with an altered cytokinin-related process.

FIELD OF THE INVENTION

The present invention is directed to plant genetic engineering. Inparticular, it relates to methods of modulating cytokinin relatedprocesses in a plant and selecting a plant having a phenotype associatedwith an altered cytokinin-related process.

BACKGROUND OF THE INVENTION

Cytokinins are a well-known class of plant growth hormones active inpromoting cell division, cell growth and differentiation, and otherphysiological processes. In particular, cytokinins are active inprocesses regulating disease resistance, stress tolerance, droughttolerance, resistance to lodging, delayed senescence, apical dominance,and assimilate partitioning in a plant, Werner et al., Proc. Natl. Acad.Sci, 98(18)10487–10492 (2001), Haberer et al., Plant Physiol., 128,pp.354–362 (2002).

Senescence, which constitutes the final phase of development in plants,is a critical stage of the plant life cycle. It is part of the agingprocess that typically occurs before cell death and is characterized bychanges in cell structure, metabolism and gene expression that effect adecline in the activities of plants. Inhibiting senescence in a planthas been identified as a way to prolong the active life-span of a plant.Certain hormones associated with senescence, e.g., cytokinin, whenpresent in increased levels in plants, have been demonstrated to delaysenescence and prolong plant activity.

It has been previously demonstrated that plants with altered senescencepatterns have leaves that retain high levels of chlorophyll throughoutseed and flower development. Tobacco plants with altered leaf senescencepatterns have enhanced yield of biomass and flower, see U.S. Pat. No.5,689,042.

Because of the importance of plants for food production, there is acontinuous and substantial effort to improve plants, e.g., create plantswith increased disease resistance phenotypes, increased stress anddrought tolerant phenotypes, increased resistance to lodging phenotypes,delayed senescence phenotypes, apical dominance phenotypes, andassimilate partitioning phenotypes. Plants with improved phenotypes arebetter able to meet the demands of food production. Accordingly, thereis a need to create plants with improved phenotypes. This inventionaddresses this and other needs.

BRIEF SUMMARY OF THE INVENTION

It has been discovered that the modulation, e.g., overexpression orunderexpression, in a plant, of a B3 domain protein will affectcytokinin related processes in the plant. Accordingly, the presentinvention provides methods of modulating cytokinin related processes ina plant. The methods of modulating a cytokinin related process in aplant comprise the following steps: (1) introducing into the plant aconstruct comprising a plant promoter operably linked to apolynucleotide wherein the polynucleotide encodes a B3 domain proteincomprising an amino acid sequence as displayed in SEQ ID NO:16, and (2)selecting a plant having a phenotype associated with an alteredcytokinin related process. In one embodiment of the present invention,the B3 domain protein comprises an amino acid sequence as displayed inSEQ ID NO:18. In a second embodiment, the B3 domain protein is SEQ IDNO:2. In a third embodiment, the B3 domain protein is SEQ ID NO:9, SEQID NO:12 or SEQ ID NO:14.

A plant promoter is used in the methods of the present invention. In oneaspect of the present invention, the plant promoter is a senescenceinducible promoter. In another aspect, the plant promoter is aconstitutive promoter, a tissue specific promoter, or a floral specificpromoter. The promoter may preferentially direct expression in ovules,pistils, anthers, fruits, seed coats, vascular tissues, provasculartissues, or apical meristems.

In one aspect of the present invention, the cytokinin related process issenescence, the phenotype selected for is delayed senescence of a plantstructure, and the selecting step comprises selecting a plant withdelayed senescence of a vegetative plant structure or a reproductiveplant structure. In one embodiment of the present invention, thevegetative structure is a leaf, stem or root. In a second embodiment,the reproductive structure is a seed, embryo, ovule, flower, pistil,anther or fruit. In a third embodiment, the selecting step comprisesselecting a plant with larger plant parts as compared to a wild typeplant, such as selecting a plant with larger seeds, larger ovules, orlarger embryos as compared to a wild type plant. In a fourth embodiment,the selecting step comprises selecting a plant with an increased numberof plant parts as compared to a wild type plant, such as selecting aplant with an increased number of seeds, an increased number of flowers,an increased number of fruits, or an increased number of stems ascompared to a wild type plant. In a fifth embodiment, the selecting stepcomprises selecting a plant with ovule development in the absence offertilization.

In another aspect of the present invention, the selecting step comprisesselecting a plant with decreased internode elongation, smaller leaves,smaller fruits or a smaller size as compared to a wild type plant.

In another aspect of the present invention, the plant promoter isoperably linked to the polynucleotide in an antisense orientation. Inyet another aspect of the present invention, the construct is introducedinto the plant by a sexual cross.

Definitions

The phrase “nucleic acid” or “polynucleotide sequence” refers to asingle or double-stranded polymer of deoxyribonucleotide orribonucleotide bases read from the 5′ to the 3′ end. Nucleic acids mayalso include modified nucleotides that permit correct read through by apolymerase and do not alter expression of a polypeptide encoded by thatnucleic acid.

The phrase “nucleic acid sequence encoding” refers to a nucleic acidwhich directs the expression of a specific protein or peptide. Thenucleic acid sequences include both the DNA strand sequence that istranscribed into RNA and the RNA sequence that is translated intoprotein. The nucleic acid sequences include both the full length nucleicacid sequences as well as non-full length sequences derived from thefull length sequences. It should be further understood that the sequenceincludes the degenerate codons of the native sequence or sequences whichmay be introduced to provide codon preference in a specific host cell.

The term “promoter” refers to a region or sequence determinants locatedupstream or downstream from the start of transcription and which areinvolved in recognition and binding of RNA polymerase and other proteinsto initiate transcription. A “plant promoter” is a promoter capable ofinitiating transcription in plant cells. Such promoters need not be ofplant origin, for example, promoters derived from plant viruses, such asthe CaMV35S promoter, can be used in the present invention.

The term “plant” includes whole plants, shoot vegetativeorgans/structures (e.g. leaves, stems and tubers), roots, flowers andfloral organs/structures (e.g. bracts, sepals, petals, stamens, carpels,anthers and ovules), seed (including embryo, endosperm, and seed coat)and fruit (the mature ovary), plant tissue (e.g. vascular tissue, groundtissue, and the like) and cells (e.g. guard cells, egg cells, trichomesand the like), and progeny of same. The class of plants that can be usedin the method of the invention is generally as broad as the class ofhigher and lower plants amenable to transformation techniques, includingangiosperms (monocotyledonous and dicotyledonous plants), gymnosperms,ferns, bryophytes, and multicellular algae. It includes plants of avariety of ploidy levels, including aneuploid, polyploid, diploid,haploid and hemizygous.

The phrase “host cell” refers to a cell from any organism. Preferredhost cells are derived from plants, bacteria, yeast, fungi, insects orother animals. Methods for introducing polynucleotide sequences intovarious types of host cells are well known in the art.

A polynucleotide sequence is “heterologous to” a second polynucleotidesequence if it originates from a foreign species, or, if from the samespecies, is modified by human action from its original form. Forexample, a promoter operably linked to a heterologous coding sequencerefers to a coding sequence from a species different from that fromwhich the promoter was derived, or, if from the same species, a codingsequence which is different from any naturally occurring allelicvariants.

A polynucleotide “exogenous to” an individual plant is a polynucleotidewhich is introduced into the plant, or a predecessor generation of theplant, by any means other than by a sexual cross. Examples of means bywhich this can be accomplished are described below, and includeAgrobacterium-mediated transformation, biolistic methods,electroporation, in planta techniques, and the like.

A nucleic acid or polynucleotide encoding a B3 domain protein is anucleic acid sequence comprising (or consisting of) a coding region ofabout 50 to about 6800 nucleotides, sometimes from about 100 to about3000 nucleotides and sometimes from about 300 to about 1300 nucleotideswhich encodes a B3 domain of about 115 amino acid residues, sometimes ofabout 105 to 125 amino acid residues, and sometimes of about 90 to about140 amino acid residues.

A “B3 domain protein” or “B3 domain polypeptide” is a protein comprisinga B3 domain. B3 domain proteins can be, e.g., sequences of about 100 toabout 1000, sometimes 200 to 450 amino acid residues. A B3 domain is asequence of about 90 to about 140, sometimes of about 105 to 125, andpreferably 115 amino acid residues. The B3 domain is a DNA bindingregion well-known and characterized in the art, see Stone et al., Proc.Natl. Acad. Sci., 98:20 11806–11811 (2001), Giraudat et al., Plant Cell,4, 1251–1261 (1992), Luerben et al., Plant J., 15, 755–764 (1998),Kagaya et al., Nucleic Acids Res. 27, 470–478 (1999), McCarty et al.,Cell, 66, 895–906, Ulmasov et al., Science, 76, 1865–1868. Examples ofproteins with B3 domains include GenBank Accession Nos: AAD20695, ARF10,CAB43843, AAF08561, ARF6, ARF8, ARF7, BIPOSTO, AAF82232, ACO25813,MP/IAA24/ARF5, ARF3/ETTIN, ARF4, ARF1, BAB10162, AAG12520, AAD20164,CAB71113, ARF9, AAF79263, AAG27097, AAD39615, AAF79371, AAF79686,AAB63625, AAD26965, AAC34233, CAB71904, AAF26476, AAC62776, BAB08947,AAF00671, RAV1, BAA95760, RAV2, ABI3, FUS3, LEC2, AAB63089, CAA16588,CAA18719, AAD20409, BAB03184, AAC69145, AAD30572, BAB02078, BAB09917,AAF29400. Exemplary embodiments of B3 domains include a B3 domainidentical or substantially identical to the B3 domain displayed in SEQID NO:7, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18.

A “LEC2 polynucleotide” is a nucleic acid sequence comprising (orconsisting of) a coding region of about 50 to about 6800 nucleotides,sometimes from about 100 to about 3000 nucleotides and sometimes fromabout 300 to about 1300 nucleotides, which hybridizes to SEQ ID NO:1under stringent conditions (as defined below), or which encodes a LEC2polypeptide or fragment of at least 15 amino acids thereof (see U.S.application Ser. No. 09/527058). LEC2 polynucleotides can also beidentified by their ability to hybridize under low stringency conditions(e.g., Tm˜40° C.) to nucleic acid probes having a the sequence of SEQ IDNO:1. SEQ ID NO:1, SEQ ID NO:5 (the LEC2 cDNA) and SEQ ID NO:6 areexamples of LEC2 polynucleotides.

A “LEC2 polypeptide” or “LEC2 protein” is a B3 domain protein. A LEC2polypeptide has a sequence of about 50 to about 400, sometimes 100 to150, and preferably 363 amino acid residues encoded by a LEC2polynucleotide. LEC2 polypeptides are plant transcription factorscharacterized by the presence of a B3 domain. For instance, amino acidresidues 158 to 272 represent the B3 domain of the polypeptide shown inSEQ ID NO:2. The B3 domain is known in the art and is shared by othertranscription factors including VIVIPAROUS1 (VP1) ((McCarty, et al.(1989) Plant Cell 1:523–532), AUXIN RESPONSE FACTOR 1 (ARF1) (Ulmasov,T. et al. (1997) Science 276:1865–1868), FUSCA3 (Luerben, H., et al.(1998) Plant J. 15:755–764) and ABI3 (Giraudat, J., et al. (1992) PlantCell 4, 1251–1261). The B3 domains of FUS3 (Reidt, W. et al. (2000)Plant J. 21:401–408), VP1 (Suzuki, M. et al. (1997) Plant Cell9:799–807) and ARF1 (Ulmasov, T., et al., supra) have been shown to beDNA binding domains. LEC2 and FUS3 both activate the promoter of astorage protein gene in transient expression assays, indicating that theB3 domain of LEC2 is a DNA binding domain and is shown in SEQ ID NO:7.

A “FUSCA3 polynucleotide” or “FUS3 polynucleotide” is a nucleic acidsequence comprising (or consisting of) a coding region of about 50 toabout 6800 nucleotides, sometimes from about 100 to about 3000nucleotides and sometimes from about 300 to about 1300 nucleotides,which hybridizes to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ IDNO:13 under stringent conditions (as defined below), or which encodes aFUS3 polypeptide or fragment of at least 15 amino acids thereof. FUS3polynucleotides can also be identified by their ability to hybridizeunder low stringency conditions (e.g., Tm˜40° C.) to nucleic acid probeshaving the sequence of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11 or SEQ IDNO:13. SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:13 areexamples of a FUS3 polynucleotide.

A “FUSCA3 polypeptide” or “FUS3 polypeptide” or “FUS3 protein” is a B3domain protein. A FUS3 polypeptide has a sequence of about 50 to about400, sometimes 100 to 300, and preferably 255 amino acid residuesencoded by a FUS3 polynucleotide. FUS3 polypeptides are planttranscription factors characterized by the presence of a B3 domain. Forinstance amino acid residues 78 to 192 represent the B3 domain of thepolypeptide shown in SEQ ID NO:9. The B3 domain of FUS3 is a DNA bindingdomain and is shown in SEQ ID NO:15.

“Increased or enhanced expression or activity of a B3 domain protein,”e.g., LEC2 or FUS3 proteins, or “increased or enhanced expression oractivity of a nucleic acid encoding a B3 domain protein,” e.g., LEC2 orFUS3 genes, refers to an augmented change in activity of the B3 domainprotein. Examples of such increased activity or expression include thefollowing: Activity of the B3 domain protein or expression of the geneencoding the B3 domain protein is increased above the level of that inwild-type, non-transgenic control plants (e.g., the quantity of LEC2 orFUS3 activity or expression of the LEC2 or FUS3 gene is increased).Activity of the B3 domain protein or expression of the gene encoding theB3 domain protein is in an organ, tissue or cell where it is notnormally detected in wild-type, non-transgenic control plants (i.e.spatial distribution of the B3 domain protein or expression of the geneencoding the B3 domain protein is altered). Activity of the B3 domainprotein or expression of the gene encoding the B3 domain protein isincreased when activity of the B3 domain protein or expression of thegene encoding the B3 domain protein is present in an organ, tissue orcell for a longer period than in a wild-type, non-transgenic controls(i.e. duration of activity of the B3 domain protein or expression of thegene encoding the B3 domain protein is increased).

“Decreased expression or activity of a B3 domain protein,” e.g., LEC2 orFUS3 proteins, or “decreased expression or activity of a nucleic acidencoding a B3 domain protein,” e.g., LEC2 or FUS3 genes, refers to adecrease in activity of the B3 domain protein. Examples of suchdecreased activity or expression include the following: Activity of theB3 domain protein or expression of the gene encoding the B3 domainprotein is decreased below the level of that in wild-type,non-transgenic control plants (e.g., the quantity of LEC2 or FUS3activity or expression of the LEC2 or FUS3 gene is decreased).

The term “reproductive structures” or “reproductive tissues” as usedherein includes fruit, ovules, seeds, pollen, flowers, or flower partssuch as pistils, stamens, sepals, petals, carpels, or any embryonictissue.

The term “vegetative structures” or “vegetative tissues” as used hereinincludes leaves, stems, tubers, roots, vascular tissue, or root andshoot meristem.

An “expression cassette” refers to a nucleic acid construct, which whenintroduced into a host cell, results in transcription and/or translationof a RNA or polypeptide, respectively. Antisense or sense constructsthat are not or cannot be translated are expressly included by thisdefinition.

In the case of both expression of transgenes and inhibition ofendogenous genes (e.g., by antisense, or sense suppression) one of skillwill recognize that the inserted polynucleotide sequence need not beidentical and may be “substantially identical” to a sequence of the genefrom which it was derived. As explained below, these variants arespecifically covered by this term.

In the case where the inserted polynucleotide sequence is transcribedand translated to produce a functional polypeptide, one of skill willrecognize that because of codon degeneracy a number of polynucleotidesequences will encode the same polypeptide. These variants arespecifically covered by the term “polynucleotide sequence from” aparticular gene, such as LEC2. In addition, the term specificallyincludes sequences (e.g., full length sequences) substantially identical(determined as described below) with a gene sequence encoding a B3domain protein, e.g., LEC2 or FUS3, and that encode proteins that retainthe function of a B3 domain protein, e.g., LEC2 or FUS3 polypeptide.

In the case of polynucleotides used to inhibit expression of anendogenous gene, the introduced sequence need not be perfectly identicalto a sequence of the target endogenous gene. The introducedpolynucleotide sequence will typically be at least substantiallyidentical (as determined below) to the target endogenous sequence.

Two nucleic acid sequences or polypeptides are said to be “identical” ifthe sequence of nucleotides or amino acid residues, respectively, in thetwo sequences is the same when aligned for maximum correspondence asdescribed below. The term “complementary to” is used herein to mean thatthe sequence is complementary to all or a portion of a referencepolynucleotide sequence.

Optimal alignment of sequences for comparison may be conducted by thelocal homology algorithm of Smith and Waterman Add. APL. Math. 2:482(1981), by the homology alignment algorithm of Needle man and Wunsch J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearsonand Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, BLAST,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 25% sequenceidentity. Alternatively, percent identity can be any integer from 25% to100%. More preferred embodiments include at least: 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 99%, compared to areference sequence using the programs described herein; preferably BLASTusing standard parameters, as described below. Accordingly, sequencesencoding a B3 domain protein used in the methods of the presentinvention include nucleic acid sequences that have substantial identityto SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, orSEQ ID NO:13. For example LEC2 sequences of the invention includenucleic acid sequences that have substantial identity to SEQ ID NO:1,SEQ ID NO:3 and SEQ ID NO:4. LEC2 sequences of the invention alsoinclude polypeptide sequences having substantial identity to SEQ IDNO:2. FUS3 sequences of the invention include nucleic acid sequencesthat have substantial identity to SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:11 or SEQ ID NO:13. FUS3 sequences of the invention also includepolypeptide sequences having substantial identity to SEQ ID NO:9, SEQ IDNO:12 or SEQ ID NO:14. One of skill will recognize that these values canbe appropriately adjusted to determine corresponding identity ofproteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning andthe like. Substantial identity of amino acid sequences for thesepurposes normally means sequence identity of at least 40%. Preferredpercent identity of polypeptides can be any integer from 40% to 100%.More preferred embodiments include at least 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. Most preferred embodimentsinclude 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74% and 75%. Polypeptides that are “substantially similar” sharesequences as noted above except that residue positions which are notidentical may differ by conservative amino acid changes. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, asparticacid-glutamic acid, and asparagine-glutamine.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other, or a third nucleic acid,under stringent conditions. Stringent conditions are sequence dependentand will be different in different circumstances. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (Tm) for the specific sequence at a defined ionic strength and pH.The Tm is the temperature (under defined ionic strength and pH) at which50% of the target sequence hybridizes to a perfectly matched probe.Typically, stringent conditions will be those in which the saltconcentration is about 0.02 molar at pH 7 and the temperature is atleast about 60° C. or 65° C.

For the purposes of this disclosure, stringent conditions forhybridizations are those which include at least one wash in 0.2×SSC at63° C. for 20 minutes, or equivalent conditions. Moderately stringentconditions include at least one wash (usually 2) in 0.2×SSC at atemperature of at least about 50° C., usually about 55° C., for 20minutes, or equivalent conditions.

The term “cytokinin related processes” refers to processes within aplant that are modulated by cytokinin. Examples of cytokinins include,but are not limited to, kinetin, zeatin, benzyl adenine. Examples ofcytokinin related processes include processes within a cell affected bycytokinin, e.g., cell division, stress tolerance, drought tolerance,disease resistance, resistance to lodging, senescence, apical dominance,and assimilate partitioning. Modulation of cytokinin related processescan result from, e.g., overproduction of cytokinin, underproduction ofcytokinin, increased sensitivity to cytokinin in a cell or decreasedsensitivity to cytokinin in a cell.

DETAILED DESCRIPTION OF THE INVENTION

A. General Overview

The present invention provides new methods of modulating cytokininrelated processes in a plant using B3 domain proteins and selecting forplants with phenotypes associated with altered cytokinin relatedprocesses. Cytokinin related processes can be modulated by overproducingcytokinin in a plant, underproducing cytokinin in a plant, increasingsensitivity to cytokinin in a plant, or decreasing sensitivity tocytokinin in a plant. The present invention is based, in part, on thesurprising discovery that increased expression of a gene that encodes aB3 domain protein, e.g., a LEC2 or FUS3 gene, in a plant inducescytokinin related processes in the plant. Cytokinin related processesinclude any process affected by cytokinin levels or activity in a plant.Examples of cytokinin related processes include, disease resistance,stress tolerance, drought tolerance, resistance to lodging, delayedsenescence, apical dominance, and assimilate partitioning.

Accordingly, the present invention provides new methods of delayingsenescence in a plant by overexpressing a B3 domain protein, e.g., aLEC2 or FUS3 protein, in the plant. The present invention also providesmethods for selecting for a plant with delayed senescence patterns orcharacteristics. Delayed senescence patterns result in plants withaltered phenotypes as compared to wild type plants. These alteredphenotypes include, but are not limited to, modulated (e.g., enhanced)size of plant parts and an increased number of plant parts. Accordingly,by overexpressing a B3 domain protein in a plant, plants with increasedbiomass and yield can be identified.

The present invention also provides methods of increasing diseaseresistance in a plant by overexpressing a B3 domain protein, e.g., aLEC2 or FUS3 protein, in the plant and selecting for a plant with anincreased disease resistance phenotype. In some embodiments, a plantwith increased disease resistance will be healthier and live longer thana wild type plant when exposed to disease conditions. Increased diseaseresistance can be measured according to any method known to those ofskill in the art. For example, disease symptoms in a test plant can becompared to disease symptoms in a control plant following contact with apathogen.

The present invention also provides methods of increasing stresstolerance in a plant by overexpressing a B3 domain protein, e.g., a LEC2or FUS3 protein, in the plant and selecting for a plant with anincreased stress tolerance phenotype. Examples of these include, e.g.,increased tolerance to drought or high salt conditions. In someembodiments, a plant with increased stress tolerance will be able toadapt better to environmental conditions as compared to a wild typeplant. For example, a plant with increased drought tolerance will haveleaves that retain their turgor in drought conditions.

The present invention also provides methods of increasing resistance tolodging in a plant by overexpressing a B3 domain protein, e.g., a LEC2or FUS3 protein, in the plant and selecting for a plant with anincreased lodging resistant phenotype. In some embodiments, a plant withincreased resistance to lodging will have thicker stems as compared to awild type plant.

B. Isolation of Nucleic Acids Used in the Methods of the PresentInvention

The isolation of sequences from the genes used in the methods of thepresent invention may be accomplished by a number of techniques. Forinstance, oligonucleotide probes based on the sequences disclosed herecan be used to identify the desired gene in a cDNA or genomic DNAlibrary from a desired plant species. To construct genomic libraries,large segments of genomic DNA are generated by random fragmentation,e.g. using restriction endonucleases, and are ligated with vector DNA toform concatemers that can be packaged into the appropriate vector. Toprepare a library of embryo-specific cDNAs, mRNA is isolated fromembryos and a cDNA library that contains the gene transcripts isprepared from the mRNA.

The cDNA or genomic library can then be screened using a probe basedupon the sequence of a cloned embryo-specific gene such as thepolynucleotides disclosed here. Probes may be used to hybridize withgenomic DNA or cDNA sequences to isolate homologous genes in the same ordifferent plant species.

Alternatively, the nucleic acids of interest can be amplified fromnucleic acid samples using amplification techniques. For instance,polymerase chain reaction (PCR) technology can be used to amplify thesequences of the genes directly from mRNA, from cDNA, from genomiclibraries or cDNA libraries. PCR and other in vitro amplificationmethods may also be useful, for example, to clone nucleic acid sequencesthat code for proteins to be expressed, to make nucleic acids to use asprobes for detecting the presence of the desired mRNA in samples, fornucleic acid sequencing, or for other purposes.

Appropriate primers and probes for identifying genes encoding a B3domain protein from plant tissues are generated from comparisons of thesequences provided herein. For a general overview of PCR see PCRProtocols: A Guide to Methods and Applications. (Innis, M, Gelfand, D.,Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990). Forexample, appropriate primers for amplification of the genomic region ofLEC2 include the following three primer pairs:D2F-5′TTTCAGAATACGCAAAAACGAC3′ (SEQ ID NO:19) andD2R-5′AACTATGCTCCCGAGTGACC3′ (SEQ ID NO:20); Ef-5′AGATGGCAAGGATCAACAGG3′(SEQ ID NO:21) and BlastR-5′CTTGCTTTCGTCCTCGTATATTG3′ (SEQ ID NO:22);and F2F-5′TTTGTGAAGCAAAATGGAGC3′ (SEQ ID NO:23) andStop-5′CGGATGAACCCACGTACG3′ (SEQ ID NO:24). Appropriate primers foramplification of the LEC2 eDNA include the following pair:5′AAATGGATAACTTCTTACCCITTCC3′ (SEQ ID NO:25) and5′CGGATGAACGCACGTACG3′(SEQ ID NO:26). The amplification conditions aretypically as follows. Reaction components: 10 mM Tris-HCl, pH 8.3, 50 mMpotassium chloride, 1.5 mM magnesium chloride, 0.00 1% gelatin, 200 μMdATP, 200 μM dCTP, 200 μM dGTP, 200 μM dTTP, 0.4 μM primers, and 100units per ml Taq polymerase. Program: 96 C for 3 min., 30 cycles of 96 Cfor 45 sec., 50 C for 60 sec., 72 for 60 sec, followed by 72 C for 5 mm.

Polynucleotides may also be synthesized by well-known techniques asdescribed in the technical literature. See, e.g., Carruthers et al.,Cold Spring Harbor Symp. Quant. Biol. 47:411–418 (1982), and Adams etal., J. Am. Chem. Soc. 105:661 (1983). Double stranded DNA fragments maythen be obtained either by synthesizing the complementary strand andannealing the strands together under appropriate conditions, or byadding the complementary strand using DNA polymerase with an appropriateprimer sequence.

The genus of nucleic acid sequences encoding B3 domain proteins used inthe methods of the present invention includes genes and gene productsidentified and characterized by analysis using the nucleic acidsequences, including SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:11 and SEQ ID NO:13 and protein sequences, includingSEQ ID NO:2, SEQ ID NO:9, SEQ ID NO:12 and SEQ ID NO:14. Sequencesencoding B3 domain proteins used in the present invention includenucleic acid sequences having substantial identity to SEQ ID NO:1, SEQID NO:5, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:13.Sequences encoding B3 domain proteins used in the present inventioninclude polypeptide sequences having substantial identity to SEQ IDNO:2, SEQ ID NO:9, SEQ ID NO:12 and SEQ ID NO:14. B3 domains used in thepresent invention include sequences having substantial identity to SEQID NO:7, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18.

The nucleic acids of the present invention encode B3 domain proteins. B3domain proteins fall into different classes or families depending uponthe relationship between their encoded B3 domains. Accordingly, in someembodiments of the present invention, the nucleic acids used in themethods of the present invention will encode a B3 domain identical orsubstantially identical to a specific class or family of B3 domainproteins, e.g., B3 domain-containing transcription factors. In someembodiments, the B3 domain-containing transcription factors bind to a RYmotif, e.g., the wild type RY motif CATGCATG, see, e.g., Reidt et al.,Plant J., 21(5), 401–408 (2000). Those of skill will recognize that B3domain proteins can be screened for the ability to bind RY motifs usingstandard assays, such as gel-shift or DNA footprinting assays, see,e.g., Maniatis et al., Molecular Cloning, Cold Spring Harbor (1982).

SEQ ID NOS:11–13 illustrate conserved B3 domain motifs. Examples 5 and 6provide alignments of B3 proteins and illustrate additional possibleamino acids in non-conserved positions. Thus, in some embodiments, B3domains comprise the amino acids that are either conserved or similar asdefined in BLAST algorithms between any of LEC2, FUS3 VP1, or asillustrated as gray boxes in Examples 5–6.

Alternatively, in some embodiments, the B3 domain-containingtranscription factors regulate embryogenesis in plants. The B3 domainproteins may be preferentially expressed in a plant cell at certaindevelopmental stages, e.g., embryogenesis. In some embodiments of thepresent invention, the nucleic acids used in the methods of the presentinvention will encode a B3 domain characteristic of the LEC2/FUS3-likeproteins. For example, in some embodiments, the B3 domain protein willcomprise a B3 domain identical or substantially identical to the B3domain found in LEC2 or FUS3. In other embodiments, the B3 domainprotein will be identical or substantially identical to the LEC2 or FUS3polypeptides as shown in SEQ ID NOS: 2 and 9. Alternatively, in someembodiments, a B3 domain protein used in the present invention will havea B3 domain characteristic of the VP1/ABI3-like proteins but will nothave other regions, e.g., masking motifs, of the protein that preventbinding with DNA. Examples of these masking motifs include amino acidresidues 1 to 491 and 632 to 659 present in the VP1 protein, see, Suzukiet al., Plant Cell, 9:799–807 (1997).

Once a nucleic acid is isolated using the method described above,standard methods can be used to determine if the nucleic acid encodes aB3 domain protein. Nucleic acids that encode B3 domain proteins can beused to create transgenic plants having delayed senescence. In someembodiments of the present invention, the B3 domain will be identical orsubstantially identical to SEQ ID NO: 7 or SEQ ID NO:15. In otherembodiments, the B3 domain will be identical or substantially identicalto the conserved regions of SEQ ID NO: 16, SEQ ID NO:17 or SEQ ID NO:18.A transgenic plant having enhanced or increased expression of a B3domain protein identical or substantially identical to SEQ ID NO:2, SEQID NO:9, SEQ ID NO:12 or SEQ ID NO:14 will display a phenotypeassociated with an altered cyotkinin process within the plant, e.g.,delayed senescence.

Alternatively, the B3 domain may be identical or substantially identicalto the LEC2 B3 domain as described in SEQ ID NO:7 or the FUS3 B3 domainas described in SEQ ID NO:15. The skilled practitioner will understandthat a nucleic acid encoding a B3 domain identical or substantiallyidentical to SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 orSEQ ID NO:18 can be used in the methods of the present invention tocreate a plant with a phenotype associated with an altered cytokininprocess with in the plant, e.g., a phenotype associated with delayedsenescence.

In other embodiments, the nucleic acid will encode a LEC2 polypeptideidentical or substantially identical to SEQ ID NO:2. Alternatively, ineven other embodiments, the nucleic acid will encode a FUS3 polypeptideidentical or substantially identical to SEQ ID NO:9, SEQ ID NO:12, orSEQ ID NO:14.

Using standard methods, the skilled practitioner can compare thesequence of a putative nucleic acid sequence thought to encode a B3domain protein to a nucleic acid sequence encoding a B3 domain proteinto determine if the putative nucleic acid encodes a B3 domain. Nucleicacids that encode a B3 domain protein, e.g., nucleic acids comprisingsequences identical or substantially identical to the B3 domains asshown in SEQ ID NOs: 7, 15, 16, 17, and 18 can be used in the methods ofthe present invention.

C. Enhancing Expression of B3 Domain Proteins

The present invention provides methods of modulating cytokinin relatedprocesses in a plant. In one embodiment of the invention, cytokininrelated processes are modulated by increasing or enhancing expression ofgene encoding a B3 domain protein in a plant, e.g., LEC2 or FUS3 genes.For example, in some embodiments, the present invention provides methodsof delaying senescence in a plant by increasing or enhancing expressionof a gene encoding a B3 domain protein in a plant, e.g., LEC2 or FUS3genes. A plant with delayed senescence possesses phenotypiccharacteristics that are recognizable to the skilled practitioner, e.g.,abnormal developmental patterns such as larger plant parts and/or anenhanced number of plants parts. The affected plant part can be areproductive plant part or vegetative plant part. For example, the plantpart may include, but is not limited to, fruit, ovules, seeds, pollen,embryonic tissue, flowers, flower parts such as pistils, stamens,sepals, petals, carpels, leaves, stems, tubers, roots, vascular tissue,provascular tissue or root or stem meristem.

In other embodiments, the present invention provides methods ofincreasing disease resistance in a plant and selecting for a plant withan increased disease resistance phenotype. A plant with increaseddisease resistance will have phenotypic characteristics that arerecognizable to the skilled practitioner, e.g., reduced symptomsfollowing exposure to a pathogen.

The nucleic acids described in the present invention may also be used toincrease stress tolerance in a plant. Accordingly, the present inventionprovides methods of increasing stress tolerance in a plant and selectinga plant with an increased stress tolerance phenotype. A plant withincreased stress tolerance will have phenotypic characteristics that arerecognizable to the skilled practitioner, e.g., increased droughttolerance.

Methods of increasing resistance to lodging in a plant or decreasingapical dominance are also embodied in the present invention.

Using specified promoters, the skilled practitioner can direct theexpression of a B3 domain protein, e.g., LEC2, and create plants withdesirable phenotypic characteristics. For example, in some embodimentsof the present invention, a tissue specific promoter, such as a seedspecific promoter, can be used to create a transgenic plant with alteredseed characteristics as compared to a wild type plant. A plant withaltered seed characteristics, for example, may have greater seed yieldor larger seeds as compared to a wild type plant. In other embodiments,the desirable characteristic may be a plant with an increased number offlowers as compared to a wild type plant. Accordingly, the skilledpractitioner may use a floral specific promoter to create a transgenicplant with the desired characteristic. Similarly, the skilledpractitioner can choose from a variety of known promoters, whetherconstitutive, inducible, tissue-specific, and the like to driveexpression of the gene encoding the B3 domain protein, e.g., LEC2 orFUS3 gene, thereby delaying senescence in a plant. Other desirablephenotypic characteristics may include leaves that stay green longer ora plant with an increased yield of fruit or an increased number ofstems.

Any phenotypic characteristic caused by alteration of cytokinin relatedprocesses in a plant, e.g., delayed senescence, can be selected for inthe present invention. For example, after introducing a polynucleotideencoding a B3 domain protein, operably linked to a desirable promoter,e.g., constitutive, tissue specific, or inducible, in a plant, andregenerating the plant by standard procedures, a skilled practitionercan use standard methods to determine if the transgenic plant is atransgenic plant of the present invention, e.g., by comparing thetransgenic plant to a wild type plant and looking for phenotypesassociated with an alteration of cytokinin related processes, e.g.,delayed senescence. In some embodiments of the present invention, theplant will be characterized by delayed ovule senescence. Delayed ovulesenescence may be evident by an ovule increased in size as compared to awild type ovule or ovule development in the absence of fertilization.

Enhancing or increasing expression of a gene encoding a B3 domainprotein in a plant may modulate cytokinin related processes by a varietyof pathways. The particular pathway used to modulate cytokinin relatedprocesses is not critical to the present invention. For example,overexpression of a B3 domain protein in a plant may affect cytokininrelated processes by increasing cytokinin levels in a plant, increasingsensitivity to cytokinin in a plant, decreasing cytokinin levels in aplant or decreasing sensitivity to cytokinin in a plant.

Any number of means well known in the art can be used to increaseactivity of a B3 domain protein, e.g., a LEC2 polypeptide, in a plant.For example, the sequences, as described herein, can be used to prepareexpression cassettes that enhance or increase endogenous geneexpression. Where overexpression of a gene is desired, the desired genefrom a different species may be used to decrease potential sensesuppression effects. Enhanced expression of polynucleotides encoding B3domains, is useful, for example, to increase the number of seedsproduced by a plant. Such techniques may be particularly useful forincreasing the yield of important plant crops.

Any organ can be targeted for overexpression of a B3 domain protein,e.g., LEC2 or FUS3, such as shoot vegetative organs/structures (e.g.,leaves, stems, and tubers), roots, flowers, and floral or reproductiveorgans/structures (e.g., bracts, sepals, petals, stamens, carpels,anthers and ovules), seed (including embryo, endosperm, and seed coat)and fruit. Vascular or provascular tissues may be targeted.Alternatively, one or several genes described in the present inventionmay be expressed constitutively (e.g., using the CaMV 35S promoter).

One of skill will recognize that the polypeptides encoded by the genesof the invention, like other proteins, have different domains whichperform different functions. Thus, the gene sequences need not be fulllength, so long as the desired functional domain of the protein isexpressed.

D. Inhibiting Expression of B3 Domain Proteins

In some embodiments of the present invention, cytokinin relatedprocesses are modulated by inhibiting gene expression in a plant. Forexample, expression cassettes of the invention can be used to suppressendogenous expression of genes encoding a B3 domain protein, e.g., FUS3or LEC2. Reducing the activity of cytokinin related processes mayincrease apical dominance, leading to less branching, or may promoteroot growth.

A number of methods can be used to inhibit gene expression in plants.For instance, antisense technology can be conveniently used. Toaccomplish this, a nucleic acid segment from the desired gene is clonedand operably linked to a promoter such that the antisense strand of RNAwill be transcribed. The expression cassette is then transformed intoplants and the antisense strand of RNA is produced. In plant cells, ithas been suggested that antisense RNA inhibits gene expression bypreventing the accumulation of mRNA which encodes the protein ofinterest, see, e.g., Sheehy et al., Proc. Natl. Acad. Sci. USA,85:8805–8809 (1988), and Hiatt et al., U.S. Pat. No. 4,801,340.

The antisense nucleic acid sequence transformed into plants will besubstantially identical to at least a portion of the endogenousembryo-specific gene or genes to be repressed. The sequence, however,does not have to be perfectly identical to inhibit expression. Thevectors of the present invention can be designed such that theinhibitory effect applies to other proteins within a family of genesexhibiting homology or substantial homology to the target gene.

For antisense suppression, the introduced sequence also need not be fulllength relative to either the primary transcription product or fullyprocessed mRNA. Generally, higher homology can be used to compensate forthe use of a shorter sequence. Furthermore, the introduced sequence neednot have the same intron or exon pattern, and homology of non-codingsegments may be equally effective. Normally, a sequence of between about30 or 40 nucleotides and about full length nucleotides should be used,though a sequence of at least about 100 nucleotides is preferred, asequence of at least about 200 nucleotides is more preferred, and asequence of at least about 500 nucleotides is especially preferred.

Transposon insertions or tDNA insertions can be used to inhibitexpression of genes, encoding B3 domain proteins. Standard methods areknown in the art. Catalytic RNA molecules or ribozymes can also be usedto inhibit expression of embryo-specific genes. It is possible to designribozymes that specifically pair with virtually any target RNA andcleave the phosphodiester backbone at a specific location, therebyfunctionally inactivating the target RNA. In carrying out this cleavage,the ribozyme is not itself altered, and is thus capable of recycling andcleaving other molecules, making it a true enzyme. The inclusion ofribozyme sequences within antisense RNAs confers RNA-cleaving activityupon them, thereby increasing the activity of the constructs.

A number of classes of ribozymes have been identified. One class ofribozymes is derived from a number of small circular RNAs that arecapable of self-cleavage and replication in plants. The RNAs replicateeither alone (viroid RNAs) or with a helper virus (satellite RNAs).Examples include RNAs from avocado sunblotch viroid and the satelliteRNAs from tobacco ringspot virus, lucerne transient streak virus, velvettobacco mottle virus, solanum nodiflorum mottle virus and subterraneanclover mottle virus. The design and use of target RNA-specific ribozymesis described in Haseloff et al. Nature, 334:585–591 (1988).

Another method of suppression is sense suppression. Introduction ofexpression cassettes in which a nucleic acid is configured in the senseorientation with respect to the promoter has been shown to be aneffective means by which to block the transcription of target genes. Foran example of the use of this method to modulate expression ofendogenous genes see, Napoli et al., The Plant Cell 2:279–289 (1990),and U.S. Pat. Nos. 5,034,323, 5,231,020, and U.S. Pat. No. 5,283,184.

Generally, where inhibition of expression is desired, some transcriptionof the introduced sequence occurs. The effect may occur where theintroduced sequence contains no coding sequence per se, but only intronor untranslated sequences homologous to sequences present in the primarytranscript of the endogenous sequence. The introduced sequence generallywill be substantially identical to the endogenous sequence intended tobe repressed. This minimal identity will typically be greater than about65%, but a higher identity might exert a more effective repression ofexpression of the endogenous sequences. Substantially greater identityof more than about 80% is preferred, though about 95% to absoluteidentity would be most preferred. As with antisense regulation, theeffect should apply to any other proteins within a similar family ofgenes exhibiting homology or substantial homology.

For sense suppression, the introduced sequence in the expressioncassette, needing less than absolute identity, also need not be fulllength, relative to either the primary transcription product or fullyprocessed mRNA. This may be preferred to avoid concurrent production ofsome plants that are overexpressers. A higher identity in a shorter thanfull length sequence compensates for a longer, less identical sequence.Furthermore, the introduced sequence need not have the same intron orexon pattern, and identity of non-coding segments will be equallyeffective. Normally, a sequence of the size ranges noted above forantisense regulation is used.

One of skill in the art will recognize that using technology based onspecific nucleotide sequences (e.g., antisense or sense suppressiontechnology), families of homologous genes can be suppressed with asingle sense or antisense transcript. For instance, if a sense orantisense transcript is designed to have a sequence that is conservedamong a family of genes, then multiple members of a gene family can besuppressed. Conversely, if the goal is to only suppress one member of ahomologous gene family, then the sense or antisense transcript should betargeted to sequences with the most variance between family members.

Another means of inhibiting gene function in a plant is by creation ofdominant negative mutations. In this approach, non-functional, mutant B3domain polypeptides, which retain the ability to interact with wild-typesubunits are introduced into a plant.

D. Preparation of Recombinant Vectors

To use isolated sequences in the above techniques, recombinant DNAvectors suitable for transformation of plant cells are prepared.Techniques for transforming a wide variety of higher plant species arewell known and described in the technical and scientific literature,e.g., Weising et al. Ann. Rev. Genet. 22:421–477 (1988). A DNA sequencecoding for the desired polypeptide, for example a cDNA sequence encodinga full length protein, will preferably be combined with transcriptionaland translational initiation regulatory sequences which will direct thetranscription of the sequence from the gene in the intended tissues ofthe transformed plant.

For example, for overexpression, a plant promoter fragment may beemployed which will direct expression of the gene in all tissues of aregenerated plant. Such promoters are referred to herein as“constitutive” promoters and are active under most environmentalconditions and states of development or cell differentiation. Examplesof constitutive promoters include the cauliflower mosaic virus (CaMV)35S transcription initiation region, the 1′- or 2′-promoter derived fromT-DNA of Agrobacterium tumefaciens, and other transcription initiationregions from various plant genes known to those of skill.

Alternatively, the plant promoter may direct expression of thepolynucleotide of the invention in a specific tissue (tissue-specificpromoters), organ (organ-specific promoters) or may be otherwise undermore precise environmental control (inducible promoters). Examples oftissue-specific promoters under developmental control include promotersthat initiate transcription only in certain tissues, such as fruit,seeds, flowers, pistils, or anthers. Suitable promoters include thosefrom genes encoding storage proteins or the lipid body membrane protein,oleosin. Examples of environmental conditions that may affecttranscription by inducible promoters include anaerobic conditions,elevated temperature, or the presence of light.

If proper polypeptide expression is desired, a polyadenylation region atthe 3′-end of the coding region should be included. The polyadenylationregion can be derived from the natural gene, from a variety of otherplant genes, or from T-DNA.

The vector comprising the sequences (e.g., promoters or coding regions)from genes of the invention will typically comprise a marker gene thatconfers a selectable phenotype on plant cells. For example, the markermay encode biocide resistance, particularly antibiotic resistance, suchas resistance to kanamycin, G418, bleomycin, hygromycin, or herbicideresistance, such as resistance to chlorosulfuron or Basta.

Nucleic acid sequences of the invention, e.g., nucleic acid sequencesthat encode B3 domain proteins, are expressed recombinantly in plantcells to enhance and increase levels of endogenous plant transcriptionfactors. For example, LEC2 or FUS3 nucleic acid sequences of theinvention are expressed recombinantly in plant cells to enhance andincrease levels of endogenous LEC2 or FUS3 polypeptides. A variety ofdifferent expression constructs, such as expression cassettes andvectors suitable for transformation of plant cells can be prepared.Techniques for transforming a wide variety of higher plant species arewell known and described in the technical and scientific literature,e.g., Weising et al. Ann. Rev. Genet. 22:421–477 (1988). A DNA sequencecoding for a polypeptide described in the present invention, e.g., acDNA sequence encoding a full length LEC2 protein, can be combined withcis-acting (promoter and enhancer) transcriptional regulatory sequencesto direct the timing, tissue type and levels of transcription in theintended tissues of the transformed plant. Translational controlelements can also be used.

The invention provides a nucleic acid encoding a B3 domain proteinoperably linked to a promoter which, in some embodiments, is capable ofdriving the transcription of the coding sequence in plants. The promotercan be, e.g., derived from plant or viral sources. The promoter can be,e.g., constitutively active, inducible, or tissue specific. Inconstruction of recombinant expression cassettes, vectors, transgenics,of the invention, different promoters can be chosen and employed todifferentially direct gene expression, e.g., in some or all tissues of aplant or animal. Typically, as discussed above, desired promoters areidentified by analyzing the 5′ sequences of a genomic clonecorresponding to the embryo-specific genes described here.

Constitutive Promoters

A promoter fragment can be employed which will direct expression of anucleic acid encoding a B3 domain protein, e.g., LEC2 or FUS3, in alltransformed cells or tissues, e.g. as those of a regenerated plant. Suchpromoters are referred to herein as “constitutive” promoters and areactive under most environmental conditions and states of development orcell differentiation. Examples of constitutive promoters include thosefrom viruses which infect plants, such as the cauliflower mosaic virus(CaMV) 35S transcription initiation region (see, e.g., Dagless (1997)Arch. Virol. 142:183–191); the 1′- or 2′-promoter derived from T-DNA ofAgrobacterium tumefaciens (see, e.g., Mengiste (1997) supra; O'Grady(1995) Plant Mol. Biol. 29:99–108); the promoter of the tobacco mosaicvirus; the promoter of Figwort mosaic virus (see, e.g., Maiti (1997)Transgenic Res. 6:143–156); actin promoters, such as the Arabidopsisactin gene promoter (see, e.g., Huang (1997) Plant Mol. Biol.33:125–139); alcohol dehydrogenase (Adh) gene promoters (see, e.g.,Millar (1996) Plant Mol. Biol. 31:897–904); ACT11 from Arabidopsis(Huang et al. Plant Mol. Biol. 33:125–139 (1996)), Cat3 from Arabidopsis(GenBank No. U43147, Zhong et al., Mol. Gen. Genet. 251:196–203 (1996)),the gene encoding stearoyl-acyl carrier protein desaturase from Brassicanapus (Genbank No. X74782, Solocombe et al. Plant Physiol. 104:1167–1176(1994)), GPc1 from maize (GenBank No. X15596, Martinez et al. J. Mol.Biol 208:551–565 (1989)), Gpc2 from maize (GenBank No. U45855, Manjunathet al., Plant Mol. Biol. 33:97–112 (1997)), other transcriptioninitiation regions from various plant genes known to those of skill. Seealso Holtorf (1995) “Comparison of different constitutive and induciblepromoters for the overexpression of transgenes in Arabidopsis thaliana,”Plant Mol. Biol. 29:637–646.

Inducible Promoters

Alternatively, a plant promoter may direct expression of the nucleicacids described in the present invention, e.g.,. nucleic acids encodinga B3 domain protein, under the influence of changing environmentalconditions or developmental conditions. Examples of environmentalconditions that may effect transcription by inducible promoters includeanaerobic conditions, elevated temperature, drought, or the presence oflight. Example of developmental conditions that may effect transcriptionby inducible promoters include senescence and embryogenesis. Suchpromoters are referred to herein as “inducible” promoters. For example,the invention incorporates the drought-inducible promoter of maize (Busk(1997) supra); the cold, drought, and high salt inducible promoter frompotato (Kirch (1997) Plant Mol. Biol. 33:897–909). Examples ofdevelopmental conditions include cell aging, and embryogenesis. Forexample, the invention incorporates the senescence inducible promoter ofArabidopsis, SAG 12, (Gan and Amasino, Science, 270:1986–1988 (1995))and the embryogenesis related promoters of LEC1 (Lotan et al., Cell,93:1195–205 (1998)), LEC2 (Stone et al., Proc. Natl. Acad. of Sci.,98:11806–11811 (2001)), FUS3 (Luerssen, Plant J. 15:755–764 (1998)),AtSERK1 (Hecht et al. Plant Physiol 127:803–816 (2001)), AGL15 (Heck etal. Plant Cell 7:1271–1282 (1995)), and BBM (BABYBOOM).

Alternatively, plant promoters which are inducible upon exposure toplant hormones, such as auxins or cytokinins, are used to express thenucleic acids of the invention. For example, the invention can use theauxin-response elements E1 promoter fragment (AuxREs) in the soybean(Glycine max L.) (Liu (1997) Plant Physiol. 115:397–407); theauxin-responsive Arabidopsis GST6 promoter (also responsive to salicylicacid and hydrogen peroxide) (Chen (1996) Plant J. 10:955–966); theauxin-inducible parC promoter from tobacco (Sakai (1996) 37:906–913); aplant biotin response element (Streit (1997) Mol. Plant MicrobeInteract. 10:933–937); and, the promoter responsive to the stresshormone abscisic acid (Sheen (1996) Science 274:1900–1902). Theinvention can also use the cytokinin inducible promoters of ARR5(Brandstatter and Kieber, Plant Cell, 10:1009–1019 (1998)), ARR6(Brandstatter and Kieber, Plant Cell, 10:1009–1019 (1998)), ARR2 (Hwangand Sheen, Nature, 413:383–389 (2001)), the ethylene responsive promoterof ERF1 (Solano et al., Genes Dev. 12:3703–3714 (1998)), and theβ-estradiol inducible promoter of XVE (Zuo et al., Plant J, 24:265–273(2000)).

Plant promoters which are inducible upon exposure to chemicals reagentswhich can be applied to the plant, such as herbicides or antibiotics,are also used to express the nucleic acids of the invention. Forexample, the maize In2-2 promoter, activated by benzenesulfonamideherbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol.38:568–577) as well as the promoter of the glucocorticoid receptorprotein fusion inducible by dexamethasone application (Aoyama, Plant J.,11:605–612 (1997)); application of different herbicide safeners inducesdistinct gene expression patterns, including expression in the root,hydathodes, and the shoot apical meristem. The coding sequence of thedescribed nucleic acids can also be under the control of, e.g., atetracycline-inducible promoter, e.g., as described with transgenictobacco plants containing the Avena sativa L. (oat) argininedecarboxylase gene (Masgrau (1997) Plant J. 11:465–473); or, a salicylicacid-responsive element (Stange (1997) Plant J. 11:1315–1324).

Tissue-Specific Promoters

Alternatively, the plant promoter may direct expression of thepolynucleotide of the invention in a specific tissue (tissue-specificpromoters). Tissue specific promoters are transcriptional controlelements that are only active in particular cells or tissues at specifictimes during plant development, such as in vegetative tissues orreproductive tissues.

Examples of tissue-specific promoters under developmental controlinclude promoters that initiate transcription only (or primarily only)in certain tissues, such as vegetative tissues, e.g., roots, leaves orstems, or reproductive tissues, such as fruit, ovules, seeds, pollen,pistils, flowers, or any embryonic tissue. Reproductive tissue-specificpromoters may be, e.g., ovule-specific, embryo-specific,endosperm-specific, integument-specific, seed and seed coat-specific,pollen-specific, petal-specific, sepal-specific, or some combinationthereof.

Suitable seed-specific promoters are derived from the following genes:MAC1 from maize (Sheridan (1996) Genetics 142:1009–1020); Cat3 frommaize (GenBank No. L05934, Abler (1993) Plant Mol. Biol. 22:10131–1038);vivparous-1 from Arabidopsis (Genbank No. U93215); atmyc1 fromArabidopsis (Urao (1996) Plant Mol. Biol. 32:571–57; Conceicao (1994)Plant 5:493–505); napA and BnCysP1 from Brassica napus (GenBank No.J02798, Josefsson (1987) JBL 26:12196–1301, Wan et al., Plant J 30:1–10(2002)); and the napin gene family from Brassica napus (Sjodahl (1995)Planta 197:264–271). Fruit specific promoters include the promoter fromthe CYP78A9 gene (Ito and Meyerowitz, Plant Cell, 12:1541–1550 (2000)).

The ovule-specific BEL1 gene described in Reiser (1995) Cell 83:735–742,GenBank No. U39944, can also be used. See also Ray (1994) Proc. Natl.Acad. Sci. USA 91:5761–5765. The egg and central cell specific FIE1promoter is also a useful reproductive tissue-specific promoter.

Sepal and petal specific promoters are also used to express nucleicacids encoding a B3 domain protein in a reproductive tissue-specificmanner. For example, the Arabidopsis floral homeotic gene APETALA1 (AP1)encodes a putative transcription factor that is expressed in youngflower primordia, and later becomes localized to sepals and petals (see,e.g., Gustafson-Brown (1994) Cell 76:131–143; Mandel (1992) Nature360:273–277). A related promoter, for AP2, a floral homeotic gene thatis necessary for the normal development of sepals and petals in floralwhorls, is also useful (see, e.g., Drews (1991) Cell 65:991–1002; Bowman(1991) Plant Cell 3:749–758). Another useful promoter is thatcontrolling the expression of the unusual floral organs (ufo) gene ofArabidopsis, whose expression is restricted to the junction betweensepal and petal primordia (Bossinger (1996) Development 122:1093–1102).

A maize pollen-specific promoter has been identified in maize (Guerrero(1990) Mol. Gen. Genet. 224:161–168). Other genes specifically expressedin pollen are described, e.g., by Wakeley (1998) Plant Mol. Biol.37:187–192; Ficker (1998) Mol. Gen. Genet. 257:132–142; Kulikauskas(1997) Plant Mol. Biol. 34:809–814; Treacy (1997) Plant Mol. Biol.34:603–611.

Promoters specific for pistil and silique valves, inflorescencemeristems, cauline leaves, and the vasculature of stem and floralpedicels include promoters from the FUL gene Mandel and Yanofsky, PlantCell, 7:1763–1771 (1995). Promoters specific for developing carpels,placenta, septum, and ovules are also used to express LEC2 nucleic acidsin a tissue-specific manner. They include promoters from the SHP1 andSHP2 genes (Flanagan et al. Plant J 10:343–353 (1996), Savidge et al.,Plant Cell 721–733). Promoters specific for the anther tapetum may bederived from the TA29 gene (Goldbeg et al., Philos Trans. R. Soc. Lond.B. Biol. Sci. 350:5–17).

Other suitable promoters include those from genes encoding embryonicstorage proteins. For example, the gene encoding the 2S storage proteinfrom Brassica napus, Dasgupta (1993) Gene 133:301–302; the 2s seedstorage protein gene family from Arabidopsis; the gene encoding oleosin20 kD from Brassica napus, GenBank No. M63985; the genes encodingoleosin A, Genbank No. U09118, and, oleosin B, Genbank No. U09119, fromsoybean; the gene encoding oleosin from Arabidopsis, Genbank No. Z17657;the gene encoding oleosin 18 kD from maize, GenBank No. J05212, Lee(1994) Plant Mol. Biol. 26:1981–1987; and, the gene encoding lowmolecular weight sulphur rich protein from soybean, Choi (1995) Mol Gen,Genet. 246:266–268, can be used. The tissue specific E8 promoter fromtomato is particularly useful for directing gene expression so that adesired gene product is located in fruits. Suitable promoters may alsoinclude those from genes expressed in vascular tissue, such as theATHB-8, AtPIN1, AtP5K1 or TED3 genes (Baima et al., Plant Physiol.126:643–655 (2001), Galaweiler et al., Science, 282:2226–2230 (1998),Elge et al., Plant J., 26:561–571 (2001), Igarashi et al., Plant Mol.Biol., 36:917–927 (1998)).

A tomato promoter active during fruit ripening, senescence andabscission of leaves and, to a lesser extent, of flowers can be used(Blume (1997) Plant J. 12:731–746). Other exemplary promoters includethe pistil specific promoter in the potato (Solanum tuberosum L.) SK2gene, encoding a pistil-specific basic endochitinase (Ficker (1997)Plant Mol. Biol. 35:425–431); the Blec4 gene from pea (Pisum sativum cv.Alaska), active in epidermal tissue of vegetative and floral shootapices of transgenic alfalfa. This makes it a useful tool to target theexpression of foreign genes to the epidermal layer of actively growingshoots.

A variety of promoters specifically active in vegetative tissues, suchas leaves, stems, roots and tubers, can also be used to express thenucleic acids used in the methods of the invention. For example,promoters controlling patatin, the major storage protein of the potatotuber, can be used, e.g., Kim (1994) Plant Mol. Biol. 26:603–615; Martin(1997) Plant J. 11:53–62. The ORF13 promoter from Agrobacteriumrhizogenes which exhibits high activity in roots can also be used(Hansen (1997) Mol. Gen. Genet. 254:337–343). Other useful vegetativetissue-specific promoters include: the tarin promoter of the geneencoding a globulin from a major taro (Colocasia esculenta L. Schott)corm protein family, tarin (Bezerra (1995) Plant Mol. Biol. 28:137–144);the curculin promoter active during taro corm development (de Castro(1992) Plant Cell 4:1549–1559) and the promoters for the tobaccoroot-specific gene TobRB7, whose expression is localized to rootmeristem and immature central cylinder regions (Yamamoto (1991) PlantCell 3:371–382).

Leaf-specific promoters, such as the ribulose biphosphate carboxylase(RBCS) promoters can be used. For example, the tomato RBCS1, RBCS2 andRBCS3A genes are expressed in leaves and light-grown seedlings, onlyRBCS1 and RBCS2 are expressed in developing tomato fruits (Meier (1997)FEBS Lett. 415:91–95). A ribulose bisphosphate carboxylase promotersexpressed almost exclusively in mesophyll cells in leaf blades and leafsheaths at high levels, described by Matsuoka (1994) Plant J. 6:311–319,can be used. Another leaf-specific promoter is the light harvestingchlorophyll a/b binding protein gene promoter, see, e.g., Shiina (1997)Plant Physiol. 115:477–483; Casal (1998) Plant Physiol. 116:1533–1538.The Arabidopsis thaliana myb-related gene promoter (Atmyb5) described byLi (1996) FEBS Lett. 379:117–121, is leaf-specific. The Atmyb5 promoteris expressed in developing leaf trichomes, stipules, and epidermal cellson the margins of young rosette and cauline leaves, and in immatureseeds. Atmyb5 mRNA appears between fertilization and the 16-cell stageof embryo development and persists beyond the heart stage. A leafpromoter identified in maize by Busk (1997) Plant J. 11:1285–1295, canalso be used.

Another class of useful vegetative tissue-specific promoters aremeristematic (root tip and shoot apex) promoters. For example, the“SHOOTMERISTEMLESS” and “SCARECROW” promoters, which are active in thedeveloping shoot or root apical meristems, described by Di Laurenzio(1996) Cell 86:423–433; and, Long (1996) Nature 379:66–69; can be used.Another useful promoter is that which controls the expression of3-hydroxy-3-methylglutaryl coenzyme A reductase HMG2 gene, whoseexpression is restricted to meristematic and floral (secretory zone ofthe stigma, mature pollen grains, gynoecium vascular tissue, andfertilized ovules) tissues (see, e.g., Enjuto (1995) Plant Cell.7:517–527). Also useful are kn1-related genes from maize and otherspecies which show meristem-specific expression, see, e.g., Granger(1996) Plant Mol. Biol. 31:373–378; Kerstetter (1994) Plant Cell6:1877–1887; Hake (1995) Philos. Trans. R. Soc. Lond. B. Biol. Sci.350:45–51. For example, the Arabidopsis thaliana KNAT1 or KNAT2promoters. In the shoot apex, KNAT1 transcript is localized primarily tothe shoot apical meristem; the expression of KNAT1 in the shoot meristemdecreases during the floral transition and is restricted to the cortexof the inflorescence stem (see, e.g., Lincoln (1994) Plant Cell6:1859–1876).

One of skill will recognize that a tissue-specific promoter may driveexpression of operably linked sequences in tissues other than the targettissue. Thus, as used herein a tissue-specific promoter is one thatdrives expression preferentially in the target tissue, but may also leadto some expression in other tissues as well.

In another embodiment, a nucleic acid described in the present inventionis expressed through a transposable element. This allows forconstitutive, yet periodic and infrequent expression of theconstitutively active polypeptide. The invention also provides for useof tissue-specific promoters derived from viruses which can include,e.g., the tobamovirus subgenomic promoter (Kumagai (1995) Proc. Natl.Acad. Sci. USA 92:1679–1683) the rice tungro bacilliform virus (RTBV),which replicates only in phloem cells in infected rice plants, with itspromoter which drives strong phloem-specific reporter gene expression;the cassava vein mosaic virus (CVMV) promoter, with highest activity invascular elements, in leaf mesophyll cells, and in root tips (Verdaguer(1996) Plant Mol. Biol. 31:1129–1139).

D. Production of Transcenic Plants

DNA constructs of the invention may be introduced into the genome of thedesired plant host by a variety of conventional techniques. For example,the DNA construct may be introduced directly into the genomic DNA of theplant cell using techniques such as electroporation and microinjectionof plant cell protoplasts, or the DNA constructs can be introduceddirectly to plant tissue using biolistic methods, such as DNA particlebombardment. Alternatively, the DNA constructs may be combined withsuitable T-DNA flanking regions and introduced into a conventionalAgrobacterium tumefaciens host vector. The virulence functions of theAgrobacterium tumefaciens host will direct the insertion of theconstruct and adjacent marker into the plant cell DNA when the cell isinfected by the bacteria.

Microinjection techniques are known in the art and well described in thescientific and patent literature. The introduction of DNA constructsusing polyethylene glycol precipitation is described in Paszkowski etal. Embo J. 3:2717–2722 (1984). Electroporation techniques are describedin Fromm et al. Proc. Natl. Acad. Sci. USA 82:5824 (1985). Biolistictransformation techniques are described in Klein et al. Nature 327:70–73(1987).

Agrobacterium tumefaciens-mediated transformation techniques, includingdisarming and use of binary vectors, are well described in thescientific literature. See, for example Horsch et al. Science233:496–498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803(1983).

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and thus the desired phenotypesuch as seedlessness. Such regeneration techniques rely on manipulationof certain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide and/or herbicide marker which has been introducedtogether with the desired nucleotide sequences. Plant regeneration fromcultured protoplasts is described in Evans et al., Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp. 124–176, MacMillilanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21–73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee etal. Ann. Rev. of Plant Phys. 38:467–486 (1987).

The nucleic acids of the invention can be used to confer desired traitson essentially any plant. Thus, the invention has use over a broad rangeof plants, including species from the genera Asparagus, Atropa, Avena,Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus,Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum,Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot,Majorana, Medicago, Nicotiana, Oryza, Panieum, Pannesetum, Persea,Pisum, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum,Sorghum, Trigonella, Triticum, Vitis, Vigna, and, Zea. The LEC2 genes ofthe invention are particularly useful in the production of transgenicplants in the genus Brassica. Examples include broccoli, cauliflower,brussel sprouts, canola, and the like.

E. Detection of the Transgenic Plants of the Present Invention

Using known procedures, one of skill can screen for plants of theinvention by detecting increased or decreased levels of B3 domainproteins in a plant and detecting the desired phenotype. Means fordetecting and quantifying mRNA or proteins are well known in the art,e.g., Northern Blots, Western Blots or activity assays. For example,after introduction of the expression cassette into a plant, the plantsare screened for the presence of the transgene and crossed to an inbredor hybrid line. Progeny plants are then screened for the presence of thetransgene and self-pollinated. Progeny from the self-pollinated plantsare grown. The resultant transgenic plants can be examined for any ofthe phenotypic characteristics associated with altered cytokinin relatedprocesses, e.g., characteristics associated with delayed senescence. Forexample, using the methods of the present invention, overexpression ofthe nucleic acids or proteins described in the present invention, e.g.,B3 domain proteins such as LEC2 or FUS3, may delay senescence in cellsof a vegetative or reproductive plant structure. The skilledpractitioner can use standard methods to determine if a plant possessesthe characteristics associated with delayed senescence. For example,leaf color can be examined to determine if the photosynthetic life-spanof the plant has been effected. Plants with extended photosynthetic lifecycles are characterized by leaves that stay green for a longer durationof time as compared to wild type plants. The size of plant vegetativeand reproductive structures can be examined to determine if they arelarger or smaller than those of a wild type plant. Transgenic plants ofthe present invention may possess larger fruit, ovules, seeds, pollen,embryonic tissue, flowers, flower parts such as pistils, stamens,sepals, petals, carpels, leaves, stems, tubers, roots, vascular tissue,provascular tissue or root or stem meristems. In other embodiments,transgenic plants of the present invention may have decreased internodeelongation, smaller leaves, smaller fruits or a smaller size as comparedto a wild type plant.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

EXAMPLES Example 1

Overexpression of LEC2

LEC2 cDNA fused with the 35S CaMV promoter was transferred into lec2-1and lec2-5 mutants and into wild type Ws-0 plants using theAgrobacterium floral dipping method. Similar overexpression phenotypeswere observed in mutant and wild type backgrounds. Fleshy embryo-like T1seedlings with unexpanded cotyledons and unextended hypocotyls andradicles were often obtained on hormone-free medium. These, as well asother more wild type looking seedlings, produced calli. Somatic embryos,cotyledon-like organs, leaves and shoots often emerged from calli. Rootswere induced less regularly, were sometimes abnormal in thickness,anatomy and color, and were sometimes ectopically induced on leaf andfloral organs. Somatic embryos readily germinated and further inducedthe production of callus, somatic embryos, and vegetative organs, whichlead to the formation of large plantlet masses. In contrast to excisedwild type leaves that senesced when cultured on hormone-free medium,35S::LEC2 leaves did not senesce, and instead induced the formation ofcalli, leaves, shoots, cotyledon-like organs, somatic embryos, andoccasionally roots. These phenotypes indicate that LEC2 is capable ofestablishing embryogenic competence in cells. In addition, ectopicexpression of LEC2 creates a proliferative organogenic environment. T1seedlings containing the 35S::LEC2 transgene with good root growth weretransferred to soil and subsequently developed into plants with smallstature, small leaves, thicker stems, limited internode elongation,reduced apical dominance, and floral abnormalities including malesterility. These 35S::LEC2 plants remained green and continued to growlong after wild type plants of the same age had died, indicating a delayin leaf and stem senescence.

One of the most striking phenotypes of 35S::LEC2 T1 plants grown on soilis the continued growth of ovules in the absence of fertilization. Wildtype ovule embryo sacs and integument cells collapsed by 10 dayspost-anthesis in the absence of fertilization. Conversely, unfertilized35S::LEC2 ovules did not senesce, and usually grew larger than wild typeseeds. Ovule growth was strictly due to integument cell division andenlargement; the embryo sac did not persist. This is the firstobservation of unfertilized, non-senescent ovules in Arabidopsis.

Pollinated 35S::LEC2 pistils developed into siliques that were shorterand wider than wild type. At 20 days after pollination, 35S::LEC2siliques remained green and non-dehiscent whereas wild type siliques hadyellowed and were beginning to dehisce. Thus, 35S::LEC2 delayed siliquesenescence. Unpollinated 35S::LEC2 pistils that enclosed the growingovules elongated and developed into structures with characteristicssimilar to 35S::LEC2 siliques. In the absence of pollination, wild typepistils elongated only slightly prior to their senescence around 10 dayspost anthesis. These results indicate that the presence of LEC2circumvents normal pistil death that occurs in the absence ofpollination and delays the senescence of the resulting fruit structures.

35S::LEC2 T1 plants that were pollinated with wild type pollen formedsiliques in which the majority of seeds were larger than wild type, andall had fleshy seed coats. Embryos within these seeds were usuallyvaried in shape, but most were larger than wild type in size. Embryosize and shape did not segregate into discrete categories, and did notappear to be associated with the presence of the transgene in theembryo. The fleshy seed coats result from continued cell divisions andthe delay in cell death that normally occurs during maturation in wildtype seed coats. Reciprocal cross experiments in which wild type plantswere pollinated with 35S::LEC2 pollen resulted in 100% wild type lookingseeds and embryos. These results indicate that LEC2 affects both seedsize and shape through its expression in maternally-derived tissues. Thedelayed senescence of the 35S::LEC2 silique allows all its seeds,regardless of whether the embryo contains the 35S::LEC2 transgene, tocontinue to grow longer than wild type and, thus, to achieve a largersize.

Taken together, the increased life span of the 35S::LEC2 whole plants,siliques, ovules, and seeds, and the lack of senescence of ovules inunpollinated pistils and excised leaves indicate that LEC2 is sufficientto delay senescence.

Example 2

Cytokinin Associated Delay in Senescence

An increase in cytokinins either by exogenous application or byincreasing endogenous levels is often associated with a delay insenescence. We used a GUS reporter gene under the control of a promoterfrom the cytokinin inducible gene, ARR5 (Agostino et al. (2000) PlantPhysiol 124:1706), to indirectly identify changes in the level of orsensitivity to cytokinins. Both wild type and 35S::LEC2 pistils atanthesis had similar levels of ARR5 regulated GUS activity in septa andfuniculi and appeared to be associated with vascular tissues. At 5 dayspost anthesis, 35S::LEC2 unpollinated pistils maintained this level ofARR5 promoter activity in septa and funiculi similar to wild typepollinated siliques, whereas wild type pistils at the same age displayedlower levels of ARR5 promoter activity in these tissues. In pollinatedsiliques at late stages of seed development, 35S::LEC2 siliquesdisplayed higher levels of ARR5 promoter activity than did wild typesiliques. These results suggest that the prolonged growth of 35S::LEC2unpollinated ovules and seeds result from delayed senescence of theovule and seeds, perhaps due to an increase in the expression ofcytokinin inducible genes.

Example 3

Overexpression of FUS3

FUS3 cDNA fused with the 35S CaMV promoter was transferred into wildtype Arabidopsis plants, ecotype Ws-0, using the Agrobacterium floraldipping method. Two types of transformed seedlings were obtained onhormone-free medium. Approximately 50% of the transformants looked likewild type seedlings except that they had slightly thicker leaves andreduced number of trichomes. The remaining 50% were in comparisondelayed in their germination and were abnormal in various ways. Aprominent abnormality was the delay in root growth. Therefore, theseseedlings were maintained on hormone-free media. Cotyledon-likestructures and fleshy leaves often grew out from the cotyledons, theshoot apical meristem and the petioles of these seedlings. Some calliwere sometimes obtained that later differentiated into stems, leaves andinflorescences and, on rarer occasions, somatic embryos. Somatic embryoswere most often formed at the margin of leaves, as well as stems andfloral organs in contact with the media. Somatic embryos germinated andgave rise to vegetative organs, calli, cotyledon-like structures, and,more rarely, somatic embryos, thus leading to the formation of plantletmasses. In contrast to excised wild type organs that senesced whencultured on hormone-free medium, 35S::FUS3 organs did not senesce, andinstead induced the formation of leaves, shoots, calli, cotyledon-likeorgans and somatic embryos. These phenotypes indicate that FUS3 issufficient to establishing embryogenic competence in cells, conferringembryonic characteristics to seedlings and inducing somatic embryoformation. FUS3 also delays senescence of plant organs. In addition,ectopic expression of FUS3 creates a proliferative, organogenicenvironment.

T1 seedlings containing the 35S::FUS3 transgene with good root growthwere transferred to soil. Most of the seedlings developed into plantswith reduced stature, limited internode elongation, lack of apicaldominance, and floral abnormalities including male sterility. Most ofthe transformants remained green and continued to grow long after wildtype plants of the same age had died, indicating a delay in leaf andstem senescence.

The transformants with the most severe lack of apical dominance anddelay in senescence showed an interesting flower phenotype: stigmaticpapilla were absent or barely started to initiate several dayspostanthesis. One interpretation of this phenotype could be a delay inthe maturation of the gynoecium. No seed were ever obtained from theseflowers because the male and female reproductive parts developedasynchronously. However, by contrast to wild type, ovules contained inthese 35S::FUS3 carpels did not senesce and degenerate. Rather, the35S::FUS3 ovules increased in size, indicating a delay in ovulesenescence and the induction of ovule cell growth and proliferation.Unpollinated 35S::FUS3 pistils that enclosed the enlarged ovuleselongated and had thicker, fleshier walls than wild type unpollinatedpistils or developing silique walls. Eventually, the fruit structuressenesced and the valves yellowed but the replum and septums usuallyremained green and fleshy for a longer period of time. These resultsindicate that the presence of FUS3 circumvents normal pistil death thatoccurs in the absence of pollination and delays the senescence of theresulting fruit structures.

Flowers sometimes reverted to a more wild type development, whichallowed fertilization and seed development. In most of the fertile T1plants, seed development occurred normally, although siliques elongationwas often reduced. The fertile 35S::FUS3 plants produced seeds that wereundistinguishable from wild type seeds in morphology and viability.

Taken together, the increased life span of the 35S::FUS3 whole plantsand the lack of senescence of ovules in unpollinated pistils and excisedorgans indicate that ectopic FUS3 expression delays senescence.

Example 4

Comparison of LEC2 and FUS3 B3 Domains

ISEQ GAP run using LEC2 and FUS3 B3 domain sequences. The B3 domains ofLEC2 and FUS3 share 50% identity and 61.7% similarity.

B3 domain nt cDNA B3 domain nt cDNA % identity FUS3 Col FUS3 Ws-0 99.71% FUS3 Col FUS3 Ler  99.71% FUS3 Ws-0 FUS3 Ler   100% LEC2 Ws-0FUS3 Ws-0 56.232% LEC2 Ws-0 FUS3 Co1 56.232% B3 domain aa B3 domain aa %identity % similarity LEC2 Ws-0 FUS3 Col 50.435% 61.739% FUS3 Col FUS3Ws-0   100%   100% FUS3 Col FUS3 Ler   100%   100%

FUS3 nucleotide sequences differ in the three Arabidopsis ecotypes.However, the polymorphisms do not cause amino acid differences withinthe B3 domain.

Example 5

Consensus Sequence for LEC2/FUS3/ABI3/VP1 B3 Domains

The following amino acid alignment of residues from the B3 domains ofLEC2 (SEQ ID NO:7), FUS3 (SEQ ID NO:15), ABI3 (SEQ ID NO:27), and VP1(SEQ ID NO:28) was created. Residues in black boxes are identical in atleast two of the four proteins, and those in the shaded boxes sharesimilarity with the conserved residues. Numbers in the right columnindicate residue numbers in the predicted polypeptides.

Example 6

Consensus Sequence for LEC2 B3 Domain Family

The following amino acid alignment of residues from the B3 domains ofAT2G30470 (SEQ ID NO:29) (GenBank Accession No. AAB63089), AT4G32010(SEQ ID NO:30) (GenBank Accession No. CAA16588), AT4G21550 (SEQ IDNO:31) (GeniBank Accession No. CAA18719), FUS3 (SEQ ID NO:32), ABI3 (SEQID NO:33) and LEC2 (SEQ ID NO:7) were created. Residues in darkly shadedboxes are identical in all six proteins. Residues in black boxes areidentical in at least three of the six proteins, and those in thelightly shaded boxes share similarity with the conserved residues.Numbers in the right column indicate residue numbers in the predictedpolypeptides. Consensus sequence=SEQ ID NO:34.

1. A method of delaying senescence, increasing the number of stems,reducing stature or increasing biomass in a plant, the methodcomprising: (i) introducing into the plant a construct comprising aplant promoter operably linked to a heterologous polynucleotide, theheterologous polynucleotide encoding a B3 domain protein comprising SEQID NO:16, and (ii) selecting a plant exhibiting, relative to a plantlacking the construct, a phenotype selected from the group consisting ofdelayed senescence, shorter stature, increased number of stems andincreased biomass.
 2. The method of claim 1, wherein the plant promoteris a constitutive promoter.
 3. The method of claim 1, wherein the plantpromoter is a tissue specific promoter.
 4. The method of claim 3,wherein the plant promoter is a floral specific promoter.
 5. The methodof claim 3, wherein the plant promoter directs expression in ovules,pistils, anthers, fruits, seed coats, vascular tissues, provasculartissues, or apical meristems.
 6. The method of claim 1, wherein theplant promoter is a senescence inducible promoter.
 7. The method ofclaim 1, wherein the selecting step comprises selecting a plantexhibiting delayed senescence, relative to a plant without theconstruct.
 8. The method of claim 7, wherein the selecting stepcomprises selecting a plant with delayed senescence in a vegetativeplant structure.
 9. The method of claim 8, wherein the vegetativestructure is a leaf, stem or root.
 10. The method of claim 7, whereinthe selecting step comprises selecting a plant with delayed senescencein a reproductive plant structure.
 11. The method of claim 10, whereinthe reproductive structure is a seed, embryo, ovule, flower, pistil,anther or fruit.
 12. The method of claim 1, wherein the selecting stepcomprises selecting a plant exhibiting shorter stature, relative to aplant without the construct.
 13. The method of claim 1, wherein theselecting step comprises selecting a plant exhibiting increased numberof stems or increased biomass, relative to a plant without theconstruct.
 14. The method of claim 1, wherein the construct isintroduced by a sexual cross.